The presence of hypoxic cells in tumors has been considered one of the main limiting factors in the control of human tumors by radiotherapy. Although a number of approaches have been proposed to overcome the hypoxic protection in radiotherapy, none of them are yet clinically useful. We found that 5-thio-D-glucose is specifically cytotoxic toward hypoxic cells. Furthermore, it sensitizes hypoxic cells to x-irradiation, whereas it protects oxic cells from radiation damage. Using four different cell lines in vitro, we demonstrated that the cytotoxicity of 5-thio-D-glucose to hypoxic cells can be dramatically enhanced by moderate hyperthermia at 41-42 degrees C. Our preliminary studies using mouse tumors also indicated that 5-thio-D-glucose can kill hypoxic cells of tumors in vivo within 1-2 hrs at 41 degrees C. This was substantiated by in vitro assay of clonogenic cells in tumors, survival of host animals, and an enhanced response of tumors to x-irradiation. Various factors may influence the effectiveness of combined use of 5-thio-D-glucose with x-irradiation. We will continue to obtain further information on the sequence of x-irradiation, 5-thio-D-glucose, and hyperthermia to obtain a favorable therapeutic ratio. This information is important for effective clinical application of 5-thio-D-glucose in conjunction with radiotherapy.